Antibody response to a fragment of the protein G of VHS rhabdovirus in immunised trout

Rocha, A.; Fernandez‐Alonso, M.; Más, Vicente; Pérez, Luis; Estepa, A.; Coll, Julio

doi:10.1016/s0165-2427(02)00016-8

Cited by 20 publications

(22 citation statements)

References 24 publications

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“…Because serum from VHSV-immunized trout strongly reacted with solid-phase frg11 (15,35) by recognizing its linear epitopes (17) and the mutants with mutations in frg11 were expressed in the cellular membrane independently of its conformation, most of the pG fusion-defective mutants described here are likely to induce immune responses in trout. Therefore, some of the mutations described in this work could be used to design attenuated VHSV vaccines, including DNA vaccination with the mutated G gene (2,3,18) or recombinant viruses obtained through reverse genetic methods (5,6).…”

Section: Discussionmentioning

confidence: 94%

“…The cells were then incubated with fluorescein isothiocyanate-conjugated goat antirabbit F(ab) 2 Transfected EPC cell-cell fusion assays. To perform the cell-cell fusion assays, EPC cells plated in 24-well plates (ϳ500,000 cells/well) were transfected with 0.6 g of the different pMCV1.4-pG mutants complexed with 2 l of Fugene (16,28,34) and incubated at 20°C. At 2 days later, the transfected cell monolayers were incubated for 15 min in RPMI Dutch culture medium plus 20 mM HEPES and 20 mM morpholineethanesulfonic acid (MES) (Sigma Chemical Co., St.Louis, Mo.)…”

Section: Methodsmentioning

confidence: 99%

“…Three of these mutations mapped around two pG locations, showing amino acid variations among VHSV isolates. Because 40% of the VHSV-immunized trout strongly recognized linear epitopes on these regions (15,17,35), the mutants described here might be useful in attempts to design attenuated VHSV strains to be used in DNA vaccines (2,3,18) or in vaccines obtained by reverse genetic methods (5,6).…”

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confidence: 99%

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Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Rocha¹,

Ruiz²,

Tafalla³

et al. 2004

J Virol

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Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.Rhabdoviruses cause highly damaging diseases in fish reared by the worldwide salmon culture industry. Among fish rhabdoviruses (novirhabdoviruses), the viral hemorrhagic septicemia virus (VHSV), which originated in Europe but has recently been found in America (26, 41), affects not only salmonids but also cod, turbot (38), sea bass, eels, flatfish, and shrimp (8,24,27). Despite many efforts, including successful DNA vaccines at the laboratory level, a commercial vaccine against VHSV is not yet available (1,18,25,30).The whole genome of VHSV has been sequenced (23), and the disulfide structure of its protein G (pG) has been elucidated (12). Reverse genetic methods have been developed for the VHSV-related rhabdovirus infectious hematopoietic necrosis virus (5-7), which could allow the design of new vaccines. The study of rhabdovirus fusion in the VHSV model could be important in the design of alternative vaccines to fight these diseases.The pG mutants of vesicular stomatitis virus (VSV), a wellstudied mammalian rhabdovirus, with an altered extent of fusion and/or optimal fusion pH in vitro, had mutations located either in the fusion peptide or in carboxy-terminal regions affecting the low-pH conformational changes required for fusion (39,40,43). Alignment of the pG sequence of VSV with those of 14 o...

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Section: Discussionmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Rocha¹,

Ruiz²,

Tafalla³

et al. 2004

J Virol

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“…Because, those results could have been due to the small size of the peptides used, trout Ab reactivity was then searched in larger G frgs. Thus the recombinant frg11 (amino acid 56e110) implicated in VHSV fusion [7], was another alternative used to estimate anti-G binding Abs [8,28].…”

Section: Introductionmentioning

confidence: 99%

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Encinas¹,

Gómez-Casado²,

Fregeneda-Grandes

et al. 2011

Fish & Shellfish Immunology

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Rainbow trout antibodies (Abs) binding to recombinant fragments (frgs) derived from the protein G of the viral haemorrhagic septicemia virus (VHSV)-07.71 strain, could be detected by ELISA (frg-ELISA) in sera from trout surviving laboratory-controlled infections. Abs were detected not only by using sera from trout infected with the homologous VHSV isolate but also with the VHSV-DK-201433 heterologous isolate, which had 13 amino acid changes. Sera from healthy trout and/or from trout surviving infectious haematopoietic necrosis virus (IHNV) infection, were used to calculate cut-off absorbances to differentiate negative from positive sera. Specific anti-VHSV Abs could then be detected by using any of the following frgs: frg11 (56-110), frg15 (65-250), frg16 (252-450) or G21-465. While high correlations were found among the ELISA values obtained with the different frgs, no correlations between any frg-ELISA and complement-dependent 50% plaque neutralization test (PNT) titres could be demonstrated. Between 4 and 10 weeks after VHSV infection, more trout sera were detected as positives by using heterologous frg-ELISA rather than homologous PNT. Furthermore, the percentage of positive sera detected by frg11-ELISA increased with time after infection to reach 100%, while those detected by complement-dependent PNT decreased to 29.4%, thus confirming that the lack of neutralizing Abs does not mean the lack of any anti-VHSV Abs in survivor trout sera. Preliminary results with sera from field samples suggest that further refinements of the frg-ELISA could allow detection of anti-VHSV trout Abs in natural outbreaks caused by different heterologous VHSV isolates. The homologous frg-ELISA method could be useful to follow G immunization attempts during vaccine development and/or to best understand the fish Ab response during VHSV infections. The viral frgs approach might also be used with other fish species and/or viruses.

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“…Conformational changes in the G protein of VHSV seem also to occur by exposure to low pH, as shown by alterations in the binding of several MAbs (Table 1) and PAbs against frg11, a region involved in fusion (12,36,40,45). Recently, a domain which regulates the low-pH-induced conformational changes has been identified both RV and VSV (19,24,35,48).…”

Section: Vol 78 2004mentioning

confidence: 98%

Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus

Más

Rocha

Pérez

et al. 2004

J Virol

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The inhibition of viral hemorrhagic septicemia rhabdovirus (VHSV) in vitro infection by pHs of <7 (low pH) has been previously reported. Nevertheless, the details of the mechanism underlying this effect remain obscure. We present evidence showing that low-pH inhibition occurs during a viral postadsorption step. Thus, while VHSV bound, replicated within single cells, and presented its G protein on the membranes of infected cells at both low and physiological pHs, both cell-to-cell spreading of infection (as estimated by the appearance of foci of infected cells) and fusion (as estimated by a syncytium assay) were inhibited by this low pH. The decreased VHSV titers and the inhibition of both cell-to-cell spreading of infection and fusion could be reversed by adjusting the pH to 7.5 at any time during infection. This effect should be taken into account to avoid false negatives in the diagnosis of VHSV by cell culture. On the other hand, the cell-to-cell spreading of infection at pH 7.5 could be stopped at any time by reducing the pH to 6.5. Since at low pH there were changes in the protein G conformation and smaller and imbalanced amounts of N with respect to M1, M2, and G viral proteins, alterations of the assembly and/or budding of VHSV are most probably involved in the absence of newly released infective virions.In many enveloped viruses, after the virus particle is internalized by a receptor-mediated endocytosis mechanism (6), viral and cell membrane fusions are triggered by the decrease of the endosomal pH (30). Conformational changes are induced by the low pH in the viral glycoproteins (25, 51) to cause fusion. Thus, agents that inhibit the lowering of the pH in the endosomes, such as NH 4 ϩ , also inhibit viral fusion and infectivity (11,44,49). However, it has been shown that a low-pH environment outside the host cells can also inhibit the in vitro viral replication and/or fusion of viruses such as vesicular stomatitis virus (VSV) (32), rabies virus (RV) (21,22,26,35), viral hemorrhagic septicemia rhabdovirus (VHSV) (10, 50, 52), influenza virus (5), poliovirus (18), or retrovirus (4,42,56).It was proposed earlier that the inhibition of VSV infectivity caused by low pH was due not to inhibition of viral RNA or protein synthesis (18) but to some yet-unidentified alterations in the nucleocapsid replication complex (32). Since then, there have been no further references about the mechanisms causing low-pH inhibition of VSV or any other rhabdoviruses, such as RV or VHSV. However, the study of this aspect of rhabdovirus infection might have important implications for the inactivation of virus in biological fluids (29) and for laboratories responsible for proper surveillance of these diseases, which should correctly diagnose possible false negatives.To further study this phenomenon, we choose VHSV (a salmonid rhabdovirus) as a model among rhabdoviruses, because VHSV causes cytopathic effects of established fish cell lines only at pH 7.0 or higher (10, 50, 52), and this might cause problems in cell culture detectio...

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Antibody response to a fragment of the protein G of VHS rhabdovirus in immunised trout

Cited by 20 publications

References 24 publications

Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Rainbow trout surviving infections of viral haemorrhagic septicemia virus (VHSV) show lasting antibodies to recombinant G protein fragments

Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus

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